Voltage sensing is ubiquitous in biology and it is fundamental in excitable tissues. This project aims at understanding voltage sensing at the molecular level. We propose to find the structural basis of function by measuring simultaneously gating and ionic currents with fluorescence changes that track structural changes in a functioning protein. The specific aims are: Aim 1: Track sensor domain conformations and interactions with the pore domain using Shaker channel as a model. Using a fluorescent arginine replacement (qBBR, either conjugated or as an unnatural amino acid), a probe we tested successfully as first gating charge in the past period, we will track the trajectory of all four gating charges during activation and deactivation at different times after activation to probe the development of the relaxed state from the active state. A newly developed physical model of the sensor will be used to interpret the results quantitatively. The studies will include non-charged residues in the S4 segment. We recently identified a non-canonical inter-subunit path of pore modulation. This path will be studied with function and fluorescence and mutagenesis and its structural basis will addressed by studying the influence of the S4 surface interaction with S5. Aim 2: Study conformational kinetics in voltage sensitive phosphatases. The trajectories of the gating residues will be studied with qBBr and site-directed fluorimetry. The extreme positions of the sensor determined with fluorimetry will be compared to the two available structures of Ci-VSP and estimation of charge displacement will be compared to charge/molecule measurements. We have characterized gating currents of ASAP1, a voltage indicator based on a V-dependent phosphatase of Gallus gallus with an inserted circularly permuted GFP, which resulted in an improved voltage sensor (ASAP1-S4). With further characterization of gating and fluorescence and, using our knowledge of other voltage sensors, we expect to develop faster and larger fluorescence change V-indicators. Because ASAP1 is much faster than Arclight (based on Ci-VSP) we plan to use it for single molecule fluorescence to obtain unitary movement of the voltage sensor. Attempts at obtaining the structure of ASAP1 to better understand the origin of fluorescence change by voltage will be done with E. Perozo. Aim 3: sodium channel Conformational changes and cooperativity. Using LRET we have obtained distance measurements in two states of the muscle Na channel, Nav1.4. We will use a homology model of Nav1.4 based on the recent structure of the cockroach Na channel to obtain detailed conformational changes with our LRET data and expand to other physical states of the channel. We will determine stoichiometry of ?1 subunit and investigate the origin of cooperativity between domains as a result of ?1 subunit in combination with clustering of channels. This research is expected to uncover voltage sensing structural basis and its adaptation to a specific function in different proteins and how particular residues may impact on mutations that cause malfunctions such as epilepsy or myotonias.